Download oil palm haploid technology: screening for naturally

OIL PALM HAPLOID TECHNOLOGY: SCREENING
FOR NATURALLY OCCURRING HAPLOIDS
647
MARIA MADON; WAN IBRAHIM WAN HASSAN; ZULKIFLI YAAKOB and
NORZIHA ABDULLAH
H
MPOB TS No. 125
MPOB INFORMATION SERIES • ISSN 1511-7871 • JUNE 2013
aploids are individuals with gametic
chromosome number (n) or having
a single set of chromosomes.
Knowledge of the haploid number
is useful in breeding and genetic
studies. Haploids are used to produce double
haploids (2n) where the chromosome number
is doubled using chemicals. Chemicals such
as colchicine or oryzalin interfere with tubulin
formation during mitosis. Double haploid (DH)
facilitate the following:
•production of completely homozygous plants
in a single generation;
•disclosure, segregation and fixation of the
genetic variation of an F1 generation;
•genetic analysis where efficient analysis and
combination of multiple (recessive) genetic
traits can be done;
•direct fixation of new target genes in a pure
line for release as new variety, especially for
self-pollinated crops;
•rapid production of F1 hybrid parent lines;
•genetic manipulation; and
•studies in genetic mapping and genomics,
such as:
a. molecular marker maps or marker assisted
breeding;
b. quantitative trait loci (QTL) mapping;
c. bulked segregation analysis; and
d. improved backcrosses.
OBJECTIVE
The objective is to screen the culled or abnormal
oil palm germinated seeds for naturally occuring
haploids via flow cytometry (FCM).
METHODOLOGY
Sorting of Abnormal Germinated Seeds
In seed production, there are at least 13 criteria
of abnormal germinated seeds. Most of these are
based on abnormalities of the plumule and radicle
(Figure 1), such as stunted plumule, radicle or
both.
Planting of Abnormal Germinated Seeds
The abnormal germinated seeds were planted in
jiffy pots according to their abnormality category
and arranged in plastic baskets on racks in the
nursery (Figure 2a). After three months, the
seedlings were analysed by FCM to determine
their ploidy level (Figure 2b).
Flow Cytometry Analysis
About 1 cm x 1 cm leaf samples of abnormal
seedlings were cut into small pieces and placed
into a 50 µm Medicon tissue homogeniser
containing LBO1 buffer supplemented with 50
µg PI (propidium iodide) and 50 µg RNaseA.
The samples were blended using Medimachine
for 3 to 5 min, filtered into a 10 ml falcon tube
and incubated at 4ºC for 24 hr followed by FCM
analysis. The fluorescence intensity of each sample
was measured by FACSCalibur flow cytometer
equipped with 15 mW argon ion laser at 488 nm.
Histograms were recorded over 1024 channels
and for leaf samples 15 000 events were captured.
The ploidy level was determined when the
haploids exhibited fluorescent histogram peak at
channel 150 -160. Finally, diploids were discarded
and haploids maintained for further confirmation
via 18S-25S ribosomal DNA fluorescent in situ
hybridisation (rDNA-FISH) or chromosome
counting.
RESEARCH FINDINGS
Screening abnormal seedlings via FCM proved to
be a useful method even though the frequency of
naturally occurring haploids was less than 0.5%.
Figure 3a shows haploid plants. Figure 3b shows
fluorescent intensity histogram peak occurring
at channel ~160 of a sample derived from a
germinated seed with stunted plumule.
Malaysian Palm Oil Board, Ministry of Plantation Industries and Commodities, Malaysia
P. O. Box 10620, 50720 Kuala Lumpur, Malaysia. Tel: 03-8769 4400
Fax: 03-8925 9446
Website: www.mpob.gov.my
Plumule
Radicle
(a) Normal germinated seeds
Stunted plumule
Colour of plumule
changed
Stunted radicle
Ratio of plumule and
radicle exceed 1:1
Radicle is too thin
Branched radicle
Growth of twin
seedlings (V shape)
Doubleton with stunted
plumule/radicle/both
Plumule and radicle in
90º position
Branched plumule
Stunted plumule and radicle
Colour of radicle or radicle
and plumule changed
(b) Abnormal germinated seeds
Figure 1. Morphology of oil palm germinated seeds: (a) normal and (b) abnormal.
Figure 2a. Abnormal germinated seeds planted in jiffy pots
arranged in plastic baskets.
a
Figure 2b. Three-month-old abnormal seedlings
(left baskets).
b
Figure 3. (a) Naturally occurring haploid plants obtained from flow cytometry screening of abnormal seedlings and
(b) fluorescent intensity histogram peak of a haploid at channel ~160.
Figure 4. Interphase nuclei exhibiting one rDNA signal (arrow) indicating the sample is a haploid.
Ribosomal DNA (18S-25S) Fluorescent in situ
Hybridisation (rDNA-FISH)
FISH of rDNA was performed on the nuclei of
putative haploids to confirm the ploidy level.
An interphase haploid nucleus will show one
major signal, diploid will exhibit two; triploid
will exhibit three major signals. Figure 4 shows
the interphase nuclei exhibiting one rDNA signal
hence indicating the sample is a haploid. The first
nucleus showed one intense signal, while the
second and third nuclei showed a diffused signal
indicating that the respective nucleolar organiser
region (NOR) was actively transcribing.
CONCLUSION
High throughput FCM is a practical method to
screen naturally occurring haploids in abnormal
oil palm germinated seeds.
For more information, kindly contact:
Director-General
MPOB
P. O. Box 10620
50720 Kuala Lumpur, Malaysia.
Tel: 03-8769 4400
Fax: 03-8925 9446
www.mpob.gov.my